U.S. Department of Health and Human

Services

National Institutes of Health

National Heart, Lung, and Blood Institute

Genotyping Automation Using Multiprobe II HT, Caliper AMS 90 SE, and Filemaker

Genotyping Automation Using Multiprobe II HT, Caliper AMS 90 SE, and Filemaker

Kaari Liisi LinaskNational Heart, Lung, and Blood InstituteKaari Liisi LinaskNational Heart, Lung, and Blood Institute

7/22/20037/22/2003

Procedure Overview

1. Sample lysis

2. Filemaker generation of CSV data files for robotics software

3. Master mix preparation

4. Multiprobe PCR setup

5. Thermal-Cycling

6. Caliper electrophoresis

Tissue Lysis

• 2mm tail biopsies are lysed in a Nunc 96 well plate with 100µL lysis buffer containing 0.2 mg/mL Proteinase K

• Incubation @ 55°C with shaking for ~4 hours on Thermomixer R with PCR plate adapter

• Heat inactivation of Proteinase K @ 95°C for 10 minutes on PTC 200 Thermocycler

Eppendorf Thermomixer R with 96-Well PCR Plate Adapters

Filemaker Genotyping Database

Five linked files:1. DNAList.fp52. MasterMix.fp53. Multiprobe.fp54. PCRList.fp55. PostMix.fp5

Convert Required Sample info into a listing of PCRs in comma-separated value format (.csv) that the Multiprobe and Caliper software can access.

Required Sample Information from 7/8/2003 DNA Plate

G20 7/8/03 A 1 Cx43 TBDG21 7/8/03 B 1 Cx43 TBDG22 7/8/03 C 1 Cx43 TBDG23 7/8/03 D 1 Cx43 TBDG24 7/8/03 E 1 Cx43 TBDG25 7/8/03 F 1 Cx43 TBDG26 7/8/03 G 1 Cx43 TBDG27 7/8/03 H 1 Cx43 TBDG28 7/8/03 A 2 Cx43 TBDG29 7/8/03 B 2 Cx43 TBDG30 7/8/03 C 2 Cx43 TBDG31 7/8/03 D 2 Cx43 TBDG32 7/8/03 E 2 Cx43 TBDG33 7/8/03 F 2 Cx43 TBDG34 7/8/03 G 2 Cx43 TBDA22 7/8/03 A 3 ETA TBDA23 7/8/03 B 3 ETA TBDR843 7/8/03 A 4 R26R TBDR844 7/8/03 B 4 R26R TBDR845 7/8/03 C 4 R26R TBDR846 7/8/03 D 4 R26R TBDR847 7/8/03 E 4 R26R TBDR848 7/8/03 F 4 R26R TBDR849 7/8/03 G 4 R26R TBDM291 7/8/03 A 5 MHC43/DHFR TBD Test Cx43 TBDM292 7/8/03 B 5 MHC43/DHFR TBD Test Cx43 TBDM293 7/8/03 C 5 MHC43/DHFR TBD Test Cx43 TBDM294 7/8/03 D 5 MHC43/DHFR TBD Test Cx43 TBDM295 7/8/03 E 5 MHC43/DHFR TBD Test Cx43 TBDM296 7/8/03 F 5 MHC43/DHFR TBD Test Cx43 TBD

1. Sample or TagID

2. PlateID (Date)

3. Row (A-H)

4. Column (1-12)

5. Genotypes To Be

Determined (Up to 3)

DNAList.fp5 Scripts

1. Import DNA Plate Records

2. Find DNA Plate Records (up to 4)

3. Assign DNA Plate# (DNA1, DNA2, DNA3, DNA4)

4. Generate Multiprobe Table

DNAList.fp5 and PCRList.fp5 files• Converts the Row (A-H) and Column (1-12) to a

Well # with Multiprobe Numbering by column• Looks up and records the PCR required for each

PCR TBD based on the PCRList.fp5

DNAList.fp5 with samples from 7/8/2003 displayed

Plate Numbering Conversions

Multiprobe:

Numbered by column

Well# = Row + (8 X Column) - 8

Caliper:Numbered by rowWell# = (12 X Row) + Column -

12

Generate Multiprobe Table Script• Deletes previous sample records from the “Multiprobe.fp5” file.• Goes through each found record’s PCR fields (1A-3B). If there is

a reaction specified, a record is added to the “Multiprobe.fp5” file for each instance with Multiprobe well #, PCR master mix required, DNA TagID, Plate#, and PlateID recorded for each PCR reaction.

• Adds (-) controls for each PCR required.• Adds `H2O` wells to fill up the last partially utilized row of the

PCR plate (required for Caliper AMS 90 SE).• Finds, sorts, and numbers the needed PCR master mixes required

including `H2O` in the “MasterMix.fp5” file.• Sorts by PCR required and TagID (ascending ASCII)• Assigns PCR plate (currently up to 2), well row, and well column

numbers arranged for Caliper AMS 90 SE processing in rows.

Multiprobe.fp5 and MasterMix.fp5 Scripts

1. Export Multiprobe Source Data

2. Print Master Mix List

3. Export Caliper Source Data

Layout for

Editing Master

Mix Recipes

Print Master Mix List Script

• Format is in the same order as it will appear on the microfuge adapter plate

• Half of the plate fits on one page

An example of samples 37 through 84 of a Multiprobe.csv file exported from Filemaker and opened in Excel

A. Master Mix SourceB. Master Mix VolumeC. Master Mix Tube #D. DNA Source PlateE. DNA VolumeF. DNA Source Well #G. PCR PlateH. PCR Well #I. # of DNA SamplesJ. PCRK. DNA TagID

Caliper CSV Table Entry Guidelines

Column A Sample Name - Text only.Column B Sample Comments - Text only.Column C Expected Fragement Base Pair Size -

Numeric only (250;720;etc.).Multiple fragments can be entered.

Column D Highlight expected fragmentsfrom Column C on electropherogram?Yes = 1, No = 0 (Default is 0).

Cells A97+ Additional notes - Text only. Start entries in Column A.

An example of the first 48 samples of a Caliper.csv file exported from Filemaker and opened in Excel

A. Concatenation of TagID and PCR

B. PCR (Master Mix)C. Expected Fragment

SizeD. Highlight Expected

Fragment Flag

MousePCR.MPT

Multiprobe II HT Deck For Multiprobe II HT Deck For Mouse Genotyping PCR Set-UpMouse Genotyping PCR Set-Up

Get DNA Sample Transfer Group

QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.

Source Plate DNA Volume DNA Well PCR Plate PCR WellDNA1 2 12 PCRPlate1 2DNA1 2 2 PCRPlate1 3DNA1 2 14 PCRPlate1 4DNA1 2 17 PCRPlate1 5DNA1 2 27 PCRPlate1 6DNA1 2 31 PCRPlate1 7DNA1 2 1 PCRPlate1 9DNA1 2 13 PCRPlate1 10

1. Get 20 µL Tips2. Using info from

source file:• Aspirate DNA• Dispense DNA

3. Drop 20 µL Tips4. Wash Tips

Close-up of Get DNA Sample Transfer Group

QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.

Source Plate DNA Volume DNA Well PCR Plate PCR WellDNA1 2 7 PCRPlate1 57DNA1 2 36 PCRPlate1 58DNA1 2 9 PCRPlate1 59DNA1 2 38 PCRPlate1 60DNA1 2 37 PCRPlate1 61DNA1 2 26 PCRPlate1 62DNA1 2 8 PCRPlate1 65DNA1 2 37 PCRPlate1 66

Get Master Mix Transfer Group

QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.

Source Volume Tube# PCRPlate PCR WellMasterMix 28 2 PCRPlate1 19MasterMix 28 2 PCRPlate1 20MasterMix 28 6 PCRPlate1 22MasterMix 28 1 PCRPlate1 25MasterMix 28 1 PCRPlate1 26MasterMix 28 2 PCRPlate1 27MasterMix 28 2 PCRPlate1 28MasterMix 28 5 PCRPlate1 29

Tip Washing

• Wash tips used after each transfer group

• Wash all tips after transfer group node loop is completed

QuickTime™ and aMotion JPEG OpenDML decompressorare needed to see this picture.

PCR Cycling Conditions

Hot-Start Program on PTC-200:1 95°C 5:00 minutes2 95°C 0:45 seconds3 58°C 0:30 seconds4 72°C 0.4°/second5 72°C 0:30 seconds6 Goto 2 39 times7 72°C 10:00 minutes8 4°C Forever9 End

Caliper Electrophoresis1. Filter Gel-Dye Mixture:

– 40 parts Gel Matrix– 1 part Fluorescent Dye Concentrate

2. Prepare DNA Ladder in PCR buffer:– 1X PCR Buffer– 2mM MgCl2

– 1X DNA Ladder3. Prepare Wash Buffer:

– 1X PCR Buffer– 2mM MgCl2

4. Prime Chip with Gel-Dye and add Markers5. Load Chip, Ladder, Wash Buffer, PCR Plate into the Caliper AMS

90 SE6. After running, rinse wells, and store chip with Storage Buffer:

– 200mM TAPS– 2mM EDTA– pH 8.0 and 0.2µM filtered

Chip Diagram from the Caliper AMS 90 SE User’s Guide

Sample Plate, Ladder, and Wash Buffer Platform from the Caliper

AMS 90 SE User’s Guide

PCR Results

• Examples of results from a single plate of PCR.

Row A Row B

Row C Row D

Row E Row F

Row G Row H

Example of a Single Lane Detail View

Adjustments and Capabilities That Have Been Added since

7/22/2003

• Adjustable master mix volumes

• Layout for addition of non-mouse database samples

• Multiple DNA source plates

• Multiple PCR plates

• Post PCR reagent addition